title: Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry.
abstract: Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually "hijack," or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses.
authors.affiliation: Institute of Molecular Biology and National RNAi Core Facility, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Su
authors.forename: Yung-Chia
authors.initials: YC
authors.lastname: Chen
authors.forename: Chung-Hsin
authors.initials: CH
authors.lastname: Tseng
authors.forename: Paul Wei-Che
authors.initials: PW
authors.lastname: Hsu
authors.forename: Kuo-Feng
authors.initials: KF
authors.lastname: Tung
authors.forename: King-Song
authors.initials: KS
authors.lastname: Jeng
authors.forename: Michael M C
authors.initials: MM
authors.lastname: Lai
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism* / Q000821:virology; D015894:Genome, Human / Q000235:genetics; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D009980:Influenza A virus / Q000254:growth & development* / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014763:Viral Matrix Proteins / Q000378:metabolism; D053586:Virus Internalization; D014779:Virus Replication
chemical_list: C488936:M1 protein, Influenza A virus; D012097:Repressor Proteins; D014763:Viral Matrix Proteins; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases